Phylogeny and systematics of the genus Calonectria

Species of Calonectria are important plant pathogens, several of which have a worldwide distribution. Contemporary taxonomic studies on these fungi have chiefly relied on DNA sequence comparisons of the β-tubulin gene region. Despite many new species being described, there has been no phylogenetic synthesis for the group since the last monographic study almost a decade ago. In the present study, the identity of a large collection of Calonectria isolates from various geographic regions was determined using morphological and DNA sequence comparisons. This resulted in the discovery of seven new species; Ca. densa, Ca. eucalypti, Ca. humicola, Ca. orientalis, Ca. pini, Ca. pseudoscoparia and Ca. sulawesiensis, bringing the total number of currently accepted Calonectria species to 68. A multigene phylogeny was subsequently constructed for all available Calonectria spp., employing seven gene regions, namely actin, β-tubulin, calmodulin, histone H3, the internal transcribed spacer regions 1 and 2 and the 5.8S gene of the ribosomal RNA, 28S large subunit RNA gene and translation elongation 1-alpha. Based on these data 13 phylogenetic groups could be distinguished within the genus Calonectria that correlated with morphological features. Dichotomous and synoptic keys to all Calonectria spp. currently recognised are also provided.Taxonomic novelties: New combinations - Calonectria angustata (Crous & El-Gholl) L. Lombard, M.J. Wingf. & Crous, Ca. australiensis (Crous & H.D. Hyde) L. Lombard, M.J. Wingf.& Crous, Ca. canadensis (J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous, Ca. chinensis (Crous) L. Lombard, M.J. Wingf. & Crous, Ca. citri (H.S. Fawc. & Klotz) L. Lombard, M.J. Wingf. & Crous, Ca. curvata (Boedijn & Reitsma) L. Lombard, M.J. Wingf. & Crous, Ca. curvispora (Crous & D. Victor) L. Lombard, M.J. Wingf. & Crous, Ca. ecuadoriae (Crous& M.J. Wingf.) L. Lombard, M.J. Wingf. & Crous, Ca. gordoniae (Leahy, T.S. Schub. & El-Gholl) L. Lombard, M.J. Wingf. & Crous, Calonectria hawksworthii (Peerally) L. Lombard, M.J. Wingf. & Crous, Calonectria hurae (Crous) L. Lombard, M.J. Wingf. & Crous, Calonectria indonesiae (Crous) L. Lombard, M.J. Wingf. & Crous, Ca. leucothoës (El-Gholl, Leahy & T.S. Schub.) L. Lombard, M.J. Wingf. & Crous, Ca. malesiana (Crous) L. Lombard, M.J. Wingf. & Crous, Ca. multiphialidica (Crous, Simoneau & Risède) L. Lombard, M.J. Wingf. & Crous, Ca. pacifica (J.C. Kang, Crous & C.L. Schoch) L. Lombard, M.J. Wingf. & Crous, Ca. penicilloides (Tubaki) L. Lombard, M.J. Wingf. & Crous, Ca. pseudonaviculata (Crous, J.Z. Groenew. & C.F. Hill) L. Lombard, M.J. Wingf. & Crous, Ca. sumatrensis (Crous) L. Lombard, M.J. Wingf. & Crous. New species - Ca. densa L. Lombard, M.J. Wingf. & Crous, Ca. eucalypti L. Lombard, M.J. Wingf. & Crous, Ca. humicola L. Lombard, M.J. Wingf. & Crous, Ca. orientalis L. Lombard, M.J. Wingf. & Crous, Ca. pini L. Lombard, M.J. Wingf. & Crous, Ca. pseudoscoparia L. Lombard, M.J. Wingf. & Crous, Ca. sulawesiensis L. Lombard, M.J. Wingf. & Crous.     

Multigene phylogeny and mating tests reveal three cryptic species related to Calonectria pauciramosa

Calonectria pauciramosa is a pathogen of numerous plant hosts worldwide. Recent studies have indicated that it included cryptic species, some of which are identified in this study. Isolates from various geographical origins were collected and compared based on morphology, DNA sequence data of the β-tubulin, histone H3 and translation elongation factor-1α regions and mating compatibility. Comparisons of the DNA sequence data and mating compatibility revealed three new species. These included Ca. colombiana sp. nov. from Colombia, Ca. polizzii sp. nov. from Italy and Ca. zuluensis sp. nov. from South Africa, all of which had distinguishing morphological features. Based on DNA sequence data, Ca. brasiliensis is also elevated to species level.Taxonomic novelties: Calonectria brasiliensis (Bat. & Cif.) L. Lombard, M.J. Wingf. & Crous, comb. nov., Calonectria colombiana L. Lombard, Crous & M.J. Wingf., sp. nov., Calonectria polizzii L. Lombard, Crous & M.J. Wingf., sp. nov., Calonectria zuluensis L. Lombard, Crous & M.J. Wingf., sp. nov.     

A multi-gene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves

Species of the ascomycete genus Mycosphaerella are regarded as some of the most destructive leaf pathogens of a large number of economically important crop plants. Amongst these, approximately 60 Mycosphaerella spp. have been identified from various Eucalyptus spp. where they cause leaf diseases collectively known as Mycosphaerella Leaf Disease (MLD). Species concepts for this group of fungi remain confused, and hence their species identification is notoriously difficult. Thus, the introduction of DNA sequence comparisons has become the definitive characteristic used to distinguish species of Mycosphaerella. Sequences of the Internal Transcribed Spacer (ITS) region of the ribosomal RNA operon have most commonly been used to consider species boundaries in Mycosphaerella. However, sequences for this gene region do not always provide sufficient resolution for cryptic taxa. The aim of this study was, therefore, to use DNA sequences for three loci, ITS, Elongation factor 1-alpha (EF-1α) and Actin (ACT) to reconsider species boundaries for Mycosphaerella spp. from Eucalyptus. A further aim was to study the anamorph concepts and resolve the deeper nodes of Mycosphaerella, for which part of the Large Subunit (LSU) of the nuclear rRNA operon was sequenced. The ITS and EF-1α gene regions were found to be useful, but the ACT gene region did not provide species-level resolution in Mycosphaerella. A phylogeny of the combined DNA datasets showed that species of Mycosphaerella from Eucalyptus cluster in two distinct groups, which might ultimately represent discrete genera.     

A taxonomic and phylogenetic revision of the Penicillium sclerotiorum complex

The morphological concept of Penicillium sclerotiorum (subgenus Aspergilloides) includes strains with monoverticillate, vesiculate conidiophores, and vivid orange to red colony colours, with colourful sclerotia sometimes produced. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region, cytochrome c oxidase subunit 1 (cox1), β-tubulin (benA), translation elongation factor 1-α (tef1-α), and calmodulin (cmd), reveal that the P. sclerotiorum morphospecies is a complex of seven phylogenetically distinct species, three of which were recently described, namely P. guanacastense, P. mallochii, and P. viticola. Three previously unidentified species are described here as P. cainii, P. jacksonii, and P. johnkrugii. The phylogenetic species are morphologically similar, but differ in combinations of colony characters, sclerotium production, conidiophore stipe roughening and branching, and conidial shape. Ecological characters and differences in geographical distribution further characterise some of the species, but increased sampling is necessary to confirm these differences. The fungal DNA barcode, the ITS, and the animal DNA barcode, cox1, have lower species resolving ability in our phylogenetic analyses, but still allow identification of all the species. Tef1-α and cmd were superior in providing fully resolved, statistically well-supported phylogenetic trees for this species complex, whereas benA resolved all species but had some issues with paraphyly. Penicilliumadametzioides and P. multicolor, considered synonyms of P. sclerotiorum by some previous authors, do not belong to the P. sclerotiorum complex. TAXONOMIC NOVELTIES: New species:Penicillium cainii K.G. Rivera, Malloch & Seifert, P. jacksonii K.G. Rivera, Houbraken & Seifert, P. johnkrugii K.G. Rivera, Houbraken & Seifert.     

石斛属植物DNA条形码序列的筛选

DNA条形码(DNA barcoding)技术是一种新的物种鉴定技术,COI序列已成功应用于动物的鉴定,但目前国际上还没有找到一个通用序列能鉴定所有植物物种。为筛选出适合于石斛属的DNA条形码序列,本文首先应用Mega 4.0软件分析了石斛属的ITS、matK、trnH-psbA、rbcL序列的特征,然后运用Wilcoxon秩和检验比较不同序列的种内和种间变异性,运用Taxon DNA软件评估这4个序列的barcoding gap。分析结果表明:ITS序列不仅种内变异和种间变异最大,而且有明显的barcoding gap,种内变异和种间变异重合较少,能较好地区分不同种类的石斛,可考虑作为石斛属DNA barcoding鉴定候选序列之一;matK、trnH-psbA和rbcL序列都不适合于石斛属DNA barcoding鉴定。在所分析的4个候选序列中,没有任何一个单一序列能完全鉴别出不同种类的石斛属植物。建议采用不同序列组合进行石斛属DNA barcoding鉴定。     

基于DNA条形码和SRAP的太子参种质遗传多样性分析

应用DNA条形码和SRAP分子标记技术,开展15份太子参种质资源遗传差异分析。太子参DNA条形码显示：ITS1序列的突变位点占比最多为2.16%,rbcL6序列的突变位点占比最少为1.23%;psbA-trnH序列缺失位点占比最多为4.88%;ITS1序列的GC含量最高,达55.52%;psbA-trnH序列的GC含量最低,仅为24.71%。太子参SRAP标记筛选出9对引物,扩增出215个位点,其中42个为多态性位点,多态性位点百分率（PPL）为19.53%;从扩增位点总数看,多态性条带丰富、清晰,既有共同位点又有特异性位点;7个居群Nei’s遗传多样性指数（H）范围为0.3466～0.4985,Shannon信息指数（I）范围为0.5301～0.6917,显示出较高的遗传多样性水平,太子参种群基因多样性（Ht）为0.4004,其中种群内基因多样性（Hs）和遗传分化系数（Gst）分别为0.0901、0.3103,分别占Ht的77.50%、22.50%。种源间Gst为0.7506,即有75.06%的遗传变异存在于种源间,24.94%的遗传变异存在于种源内,表明太子参种源间遗传变异大于种源...     

European species of Hypocrea: Part I. The green-spored species

At present 75 species of Hypocrea have been identified in temperate Europe. Nineteen green-spored species and their Trichoderma asexual states are here described in detail. Extensive searches for Hypocrea teleomorphs in 14 European countries, with emphasis on Central Europe, yielded more than 620 specimens within five years. The morphology of fresh and dry stromata was studied. In addition, available types of species described from Europe were examined. Cultures were prepared from ascospores and used to study the morphology of cultures and anamorphs, to determine growth rates, and to extract DNA that was used for amplification and sequencing of three genetic markers. ITS was used for identification, while RNA polymerase II subunit b (rpb2) and translation elongation factor 1 alpha (tef1) were analyzed for phylogenetic reconstruction of the genus.Several unexpected findings resulted from this project: 1) The previous view that only a small number of Trichoderma species form a teleomorph is erroneous. 2) All expectations concerning the number of species in Europe are by far exceeded. Seventy-five species of Hypocrea, two species of Protocrea, and Arachnocrea stipata, are herein identified in temperate Europe, based on the ITS identification routine using fresh material, on species described earlier without molecular data and on species recently described but not collected during this project. 3) Current data suggest that the biodiversity of Hypocrea / Trichoderma above soil exceeds the number of species isolated from soil. 4) The number of Trichoderma species forming hyaline conidia has been considered a small fraction. In Europe, 26 species of those forming teleomorphs produce hyaline conidia, while 42 green-conidial species are known. Three of the detected Hypocrea species do not form an anamorph in culture, while the anamorph is unknown in four species, because they have never been cultured.This work is a preliminary account of Hypocrea and their Trichoderma anamorphs in Europe. Of the hyaline-spored species, H. minutispora is by far the most common species in Europe, while of the green-spored species this is H. strictipilosa.General ecology of Hypocrea is discussed. Specific associations, either with host fungi or trees have been found, but the majority of species seems to be necrotrophic on diverse fungi on wood and bark.The taxonomy of the genus will be treated in two parts. In this first part 19 species of Hypocrea with green ascospores, including six new teleomorph and five new anamorph species, are described in detail. All green-spored species belong to previously recognised clades, except H. spinulosa, which forms the new Spinulosa Clade with two additional new species, and H. fomiticola, which belongs to the Semiorbis Clade and forms effuse to large subpulvinate stromata on Fomes fomentarius, a trait new for species with green ascospores. Anamorph names are established prospectively in order to provide a basis for possible policy alterations towards their use for holomorphs.Taxonomic novelties: Hypocrea aeruginea Jaklitsch, Trichoderma aerugineum Jaklitsch, T. dacrymycellum Jaklitsch, H. danica Jaklitsch, H./T. fomiticola Jaklitsch, H. longipilosa Jaklitsch, H./T. parepimyces Jaklitsch, H. parestonica Jaklitsch, T. parestonicum Jaklitsch.     

干旱胁迫下植物生理生化及DNA甲基化的研究进展

干旱是影响植物正常生长发育的一个最重要的逆境因子,植物为适应和抵御干旱环境所引起的生理生化过程是一个复杂的系统,DNA甲基化在植物干旱胁迫下过程中起了重要作用。从植物形态指标的表现、生理生化水平的调节及DNA甲基化3个方面归纳阐述植物对干旱胁迫的响应,综述植物抗旱响应对策、DNA甲基化及其检测方法等研究进展,为抗旱种质的鉴定、筛选和抗旱生理育种提供参考。     

Diversity in nuclear DNA content and ploidy level of Hedychium species and hybrids

Hedychiums are multipurpose plants cultivated as ornamentals because of their multicolor and showy and scented flowers and as medicinal plants because of their essential oils that have been found to possess antimicrobial and insecticidal properties. There is often taxonomic and botanical confusion about Hedychium species and cultivars because of the apparent existence of more than one cytotype within several Hedychium species, most likely because of the ease with which these species hybridize. Nuclear DNA content (2C-DNA) was determined for the first time in 23 Hedychium species and hybrids. The 2C-values ranged from 1.70 pg to 3.98 pg. We also determined the chromosome number of one species, Hedychium stenopetalum, which was found to be diploid (2n = 2x = 34), supporting the widely held view that the basic chromosome number in Hedychium is x = 17, and the existence of different cytotypes is most probably attributable to natural hybridization. Our results should be helpful not only in clarifying the botanical and taxonomic confusion in Hedychium but also in paving the way for a sound breeding program for this increasingly important ornamental ginger.     

Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders,three new families,eight new genera and one hundred and seven new species

Nearly 500 basidiomycetous yeast species were accepted in the latest edition of The Yeasts: A Taxonomic Study published in 2011. However, this number presents only the tip of the iceberg of yeast species diversity in nature. Possibly more than 99 % of yeast species, as is true for many groups of fungi, are yet unknown and await discovery. Over the past two decades nearly 200 unidentified isolates were obtained during a series of environmental surveys of yeasts in phyllosphere and soils, mainly from China. Among these isolates, 107 new species were identified based on the phylogenetic analyses of nuclear ribosomal DNA (rDNA) [D1/D2 domains of the large subunit (LSU), the small subunit (SSU), and the internal transcribed spacer region including the 5.8S rDNA (ITS)] and protein-coding genes [both subunits of DNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB)], and physiological comparisons. Forty-six of these belong to 16 genera in the Tremellomycetes (Agaricomycotina). The other 61 are distributed in 26 genera in the Pucciniomycotina. Here we circumscribe eight new genera, three new families and two new orders based on the multi-locus phylogenetic analyses combined with the clustering optimisation analysis and the predicted similarity thresholds for yeasts and filamentous fungal delimitation at genus and higher ranks. Additionally, as a result of these analyses, three new combinations are proposed and 66 taxa are validated.     

桐花树内生细菌动态及拮抗植物病原菌菌株筛选鉴定

以荧光显微计数法和平板分离法分别对桐花树根茎叶内生细菌总量和可培养细菌数量进行周年动态初步测定,并对拮抗植物病原菌的可培养细菌菌株进行筛选鉴定。结果表明:桐花树根茎叶内均含有大量的内生细菌,其中根部内生细菌总量为0.88×107~49.67×107ind./g FW,7月份最大,叶部细菌总量为1.07×107~65.07×107ind./g FW,也以7月份最大,茎部细菌总量为1.1×107~19.73×107ind./g FW,以5月份最大;而根茎叶可培养细菌含量分别为1.02×103~13.18×103、0.13×103~11.24×103、0.31×103~10.36×103CFU/g FW,均以3月份数量最大,周年不同月份之间细菌总量和可培养细菌数量均差异显著。用平板对峙生长法,从86株可培养内生细菌中筛选获得1株对香蕉叶鞘腐败病菌、香蕉枯萎病菌、马拉巴栗茎腐病等植物病原菌均有较强拮抗作用的Aec23菌株,经形态、生理生化测试及16S rDNA序列比较分析,Aec23菌株初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。     

橡胶树多主棒孢病菌Cassiicolin基因条形码数据库构建及分子检测技术

多主棒孢（Corynespora cassiicola）是为害我国主要热带作物的植物病原真菌,其寄主范围广、形态差异显著、症状类型多样,且含有一种寄主专化性毒素Cassiicolin,存在6种毒素类型。本研究利用已公布的Cassiicolin毒素基因（Cas1~Cas6型）,构建了含287个菌株的国内橡胶树和部分热带作物多主棒孢Cassiicolin基因条形码数据库,建立了一套特异性强、灵敏度高的分子检测技术,可检测100 pg/μL的目标基因组DNA,系统分析了我国橡胶树、木薯、番木瓜、瓜菜等主要热带作物的919株多主棒孢的毒素类型,发现国内仅存在Cas2和Cas5这2种毒素类型,其中Cas5型的菌株占94.8%,为橡胶树多主棒孢的优势种群和特有毒素类型。而Cas2型是橡胶树和其他作物多主棒孢共有的毒素型。系统发育分析发现,不同毒素类型的多主棒孢菌株与寄主来源密切相关,但与地理来源没有明显的相关性,且Cas5型多主棒孢具有明显的寄主专化性。通过构建多主棒孢Cassiicolin基因条形码数据库,为明确我国主要热带作物多主棒孢病菌的种群结构和优势种群情况,发掘、保存多主棒孢菌种资源以及制定病害的防治策略上具有重要的指导意义。     

一种利用菠萝蜜叶片提取高质量DNA 的方法

以菠萝蜜的干胞和湿胞类型的叶片为试材,用CTAB法初提DNA后,经过苯酚-氯仿纯化,并在高盐条件下4℃盐析、离心去多糖,乙醇沉淀,成功地从菠萝蜜叶片中提取出了可用于PCR扩增和限制性酶切分析的高质量DNA,产量在300 ugk 左右。     

中国海南海岛小花蝽(Orius maxidentex Ghauri)的发现及生物学记述

花蝽是一类捕食性半翅目昆虫。花蝽科目前约包含70个属,其中小花蝽属（Orius Wolff, 1811）是最大的属,全世界包含约80种,中国目前有15种。小花蝽属的种类在蓟马的生物防治方面具有极大的应用前景,是生物防治领域研究和利用的热门天敌类群。目前在我国针对蓟马的有害生物综合治理及绿色防控项目中,能够应用的仅东亚小花蝽（O. sauteri）及南方小花蝽（O. strigicollis）。最近在海南发现的海岛小花蝽（Orius maxidentex Ghauri）为中国新记录种,国外分布于泰国、印度、巴基斯坦、伊朗、阿拉伯联合酋长国、苏丹、塞内加尔。本研究对海岛小花蝽的形态学特征和生物学特征进行了描述,拍摄了海岛小花蝽各虫态及生殖器特征图,并对其发育历期、捕食、交配、产卵、孵化、蜕皮、羽化及同类相食的现象进行了简单描述和研究。此外,本研究获得了海岛小花蝽线粒体细胞色素氧化酶亚基I（COI）基因序列,并提交到美国国家生物技术信息中心（GenBank登录号：OL798096）进行序列比对,分子鉴定结果表明与塞内加尔的海岛小花蝽序列高度一致。海岛小花蝽常见于青葙（Celosia argentea）、小蓬草（Conyza canadensis）等杂草,及芒果（Mangifera indica）、豇豆（Vigna unguiculata）和茄子（Solanum melongena）等作物的花及幼叶上。卵产于植物组织中,若虫阶段有5个龄期,成虫和若虫均以蓟马为食,可捕食花蓟马（Frankliniella intonsa）、黄胸蓟马（Thrips hawaiiensis）、棕榈蓟马（T. palmi）、豆大蓟马（Megalurothrips usitatus）、茶黄蓟马（Scirtothrips dorsalis）及美洲棘蓟马（Echinothrips americanus）等。海岛小花蝽在海南岛全年可见,且具有明显的种群优势,是海南蓟马害虫的重要天敌。本研究在实验室条件下,利用美洲棘蓟马（E. americanus）和大豆幼苗进行室内饲养和观察,发现海岛小花蝽在温度(25±2)℃及相对湿度75%±5%条件下,可以取食美洲棘蓟马完成世代发育,卵期(3.66±0.67)d,1龄若虫(1.72±0.65)d,2龄若虫(2.21±0.82)d,3龄若虫(2.62±0.62)d,4龄若虫(2.45±0.74)d,5龄若虫(3.21± 0.82)d。本研究凭证标本保存于中国热带农业科学院环境与植物保护研究所和中国农业大学植物保护学院昆虫标本馆。     

高粱DNA提取纯化方法的比较及RAPD反应条件的建立与优化

以高粱叶片为试材，采用4种方法提取DNA，并对这4种方法进行比较，选择高粱DNA提取纯化的最佳方法，同时对高粱RAPD反应条件进行研究，从而建立了一个适合高粱RAPD分析的最佳反应系统。     

一种快速提取玉米基因组DNA的方法

实验以SDS为主要提取试剂,建立了用于玉米叶片基因组DNA的微量快速提取方法。结果表明:利用此方法提取的玉米叶片基因组DNA具有使用药品少、操作简单、迅速、成本低等优点,每人每工作日可以提取180～250个DNA样品,一个样品可供80～100次PCR反应使用。此DNA提取方法可有效用于玉米的转基因成分检测。     

植物功能基因组学研究进展

对植物功能基因组学的研究方法进行了论述,包括微阵列或DNA芯片、EST技术、SAGE技术、蛋白质组学技术、反向遗传技术及生物信息学技术等,对植物功能基因组学研究中可能遇到的问题进行了分析。     

玉米DNA指纹数据库建库标准规范的建立

为了保证玉米品种DNA指纹库构建的标准化,从建库标记、检测平台、试剂、样品、评估程序、数据整合、模式库、扩展库和随机盲测9个方面进行了全面的规范,从而为科研单位合作开展大规模的DNA指纹库构建研究奠定基础。     

假臭草DNA提取及RAPD反应体系的优化

以假臭草叶片为材料,对影响其随机扩增多态DNA(RAPD)反应的各因素进行优化,建立了假臭草RAPD的优化反应体系和程序,即在10 μL反应体系中,5 ng(/10 μL)模板DNA,1.0 μmol/L随机引物F15,150 μmol/L dNTPs,2.0 mmol/L Mg2+,1.0 U Taq DNA聚合酶;扩增程序为95℃预变性4 min,95℃变性40 s,36℃退火40 s,72℃延伸1 min,10个循环,后94℃变性30 s,35℃退火30 s,72℃延伸1 min,35个循环,72℃     

一种适于PCR扩增的荞麦DNA大量提取及纯化方法

为满足对大批量材料进行分子标记检测的需要,以荞麦叶片为材料,针对荞麦叶片中黄酮含量高,提取荞麦基因组DNA时采用改良的CTAB方法,该方法提取的DNA经琼脂糖凝胶电泳和检测,获得的DNA条带较亮、无RNA、无拖尾现象;经分光光度计检测获得数据证明酚类、蛋白质较少。利用ISSR引物对提取的荞麦基因组DNA进行PCR检测,能获得清晰稳定的条带,说明该方法提取的荞麦基因组DNA能满足PCR反应的需要。